Development of Multiple Antibiotic Resistance in Microbes (Microbial Genetics)

Multidrug Resistance in Bacteria

Hiroshi NikaidoDepartment of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202

AbstractLarge amounts of antibiotics used for human therapy, as well as for farm animals and even for fishin aquaculture, resulted in the selection of pathogenic bacteria resistant to multiple drugs. Multidrugresistance in bacteria may be generated by one of two mechanisms. First, these bacteria mayaccumulate multiple genes, each coding for resistance to a single drug, within a single cell. Thisaccumulation occurs typically on resistance (R) plasmids. Second, multidrug resistance may alsooccur by the increased expression of genes that code for multidrug efflux pumps, extruding a widerange of drugs. This review discusses our current knowledge on the molecular mechanisms involvedin both types of resistance.

KeywordsR plasmids; transposons; integrons; type IV secretion system; multidrug efflux pumps

INTRODUCTIONThe discovery of penicillin in 1928 was followed by the discovery and commercial productionof many other antibiotics. We now take for granted that any infectious disease is curable byantibiotic therapy. Antibiotics are manufactured at an estimated scale of about 100,000 tonsannually worldwide, and their use had a profound impact on the life of bacteria on earth. Morestrains of pathogens have become antibiotic resistant, and some have become resistant to manyantibiotics and chemotherapeutic agents, the phenomenon of multidrug resistance.

Indeed, some strains have become resistant to practically all of the commonly available agents.A notorious case is the methicillin-resistant Staphylococcus aureus (MRSA), which is resistantnot only to methicillin (which was developed to fight against penicillinase-producing S.aureus) but usually also to aminoglycosides, macrolides, tetracycline, chloramphenicol, andlincosamides. Such strains are also resistant to disinfectants, and MRSA can act as a majorsource of hospital-acquired infections. An old antibiotic, vancomycin, was resurrected fortreatment of MRSA infections. However, transferable resistance to vancomycin is now quitecommon in Enterococcus and found its way finally to MRSA in 2002, although such strainsare still rare (1).

An even more serious threat may be the emergence of gram-negative pathogens that areresistant to essentially all of the available agents (2). Research had time to react against thethreat by MRSA. Thus, there are newly developed agents that are active against vancomycin-resistant MRSA, such as linezolid and quinupristin/dalfopristin. However, the emergence of

Copyright © 2009 by Annual Reviews. All rights [email protected] .DISCLOSURE STATEMENT The author is not aware of any biases that might be perceived as affecting the objectivity of this review.

NIH Public AccessAuthor ManuscriptAnnu Rev Biochem. Author manuscript; available in PMC 2010 March 16.

Published in final edited form as:Annu Rev Biochem. 2009 ; 78: 119–146. doi:10.1146/annurev.biochem.78.082907.145923.

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“pan-resistant” gram-negative strains, notably those belonging to Pseudomonas aeruginosaand Acinetobacter baumanii, occurred more recently, after most major pharmaceuticalcompanies stopped the development of new antibacterial agents. Hence, there are almost noagents that could be used against these strains, in which an outer membrane barrier of lowpermeability and an array of efficient multidrug efflux pumps are combined with multitudesof specific resistance mechanisms.

Multidrug resistance in bacteria occurs by the accumulation, on resistance (R) plasmids ortransposons, of genes, with each coding for resistance to a specific agent, and/or by the actionof multidrug efflux pumps, each of which can pump out more than one drug type.

BIOCHEMICAL MECHANISMS OF RESISTANCEIt is first necessary for us to review briefly the commonly encountered mechanisms ofresistance.

Mutational Alteration of the Target ProteinMan-made compounds, such as fluoroquinolones, are unlikely to become inactivated by theenzymatic mechanisms described below. However, bacteria can still become resistant throughmutations that make the target protein less susceptible to the agent. Fluoroquinolone resistanceis mainly (but not exclusively) due to mutations in the target enzymes, DNA topoisomerases(3). Whether resistance of this type is easily transferred to other cells on plasmids depends onthe mode of the drug's action. With fluoroquinolones, which will kill the bacterial cellcontaining any drug-susceptible enzyme, the addition of the gene coding for a drug-resistantenzyme will not make the bacteria completely resistant, and plasmid-mediated transfer of themutated target gene is unlikely to occur. Nevertheless, the mutants will become more and moreprevalent by clonal selection in the presence of selective pressure.

When high-level streptomycin resistance is selected in Escherichia coli in the laboratory,mutation in one of the ribosomal proteins (RpsL) is usually selected. Again, bringing in thismutant gene on a plasmid will not make the recipient resistant because the drug action on thesusceptible ribosome (containing the chromosomally coded intact RpsL) will result in celldeath. This type of resistance is almost never found in the streptomycin-resistant E. coli ofclinical origin, presumably because resistance caused by the drug modification (see below) iseffectively expressed on plasmids. However, ribosomal resistance mutation is often found inthe aminoglycoside-resistant clinical strains of Mycobacterium tuberculosis.

Another example of resistance attributable to target modification is that conferred by the ermgene, which is usually plasmid coded and produces the methylation of adenine at position 2058of the 50S rRNA, causing resistance to macrolides (erythromycin and many others),lincosamide, and streptogramin of group B, the MLS phenotype (4). The molecular basis ofthis phenotype was elucidated through the crystal structure of 50S ribosomal subunit (5).

Sulfa drugs (synthetic competitors of p-aminobenzoic acids that inhibit dihydropteroatesynthetase and trimethoprim, a synthetic inhibitor of dihydrofolate reductase) have been usedin combination. They select for drug-resistant mutants of the respective enzymes. In this case,the high-level production of drug-resistant target enzymes from plasmids can make the bacteriaresistant, and the resistant genes have spread widely on plasmids (6).

Enzymatic Inactivation of the DrugThis is a common resistance mechanism for antibiotics of natural origin, such asaminoglycosides (kanamycin, tobramycin, and amikacin), which are inactivated by enzymaticphosphorylation [by aminoglycoside phosphoryltransferase (APH)], acetylation [by

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aminoglycoside acetyltransferase (AAC)], or adenylation (by aminoglycosideadenyltransferase or nucleotidyltransferase), and β-lactams (penicillins, cephalosporins, andcarbapenems such as imipenem), which are inactivated by enzymatic hydrolysis by β-lactamases, usually in the periplasm. Genes coding for these inactivating enzymes can easilyproduce resistance as additional genetic components on plasmids.

Aminoglycosides—Aminoglycosides are inactivated by modifications that reduce the netpositive charges on these polycationic antibiotics (7,8). There are now many dozens ofaminoglycoside-modifying enzymes known; for example, AAC (3)-II designates anaminoglycoside acetyltransferase acting on position 3 of the substrate and belonging to thesecond phylogenic grouping among these enzymes (9). It was proposed in 1973 (10) that amajor source of antibiotic-inactivating enzymes is the antibiotic-producing microorganisms,which must protect themselves. Indeed APH (3′)-I, a plasmid-coded aminoglycosidephosphorylase, which is present in 46% of the aminoglycoside-resistant gram-negativebacteria, is strongly related to a chromosomally coded enzyme in the producing strainStreptomyces fradiae (9). A similar situation also exists for AAC (3)-II, a plasmid-codedenzyme present in more than 60% of aminoglycoside-resistant gram-negative bacteriaexamined (9).

After these findings, the pharmaceutical industry developed either semisynthetic or naturalaminoglycosides that are not the substrates of common inactivating enzymes. Nevertheless,these drugs are still susceptible to the action of some enzymes, and plasmids found inaminoglycoside-resistant strains in recent years often contain genes coding for severalinactivating enzymes (11).

β-Lactams—Only a few years after the introduction of penicillin into clinical practice, S.aureus developed resistance caused by a β-lactamase coded for it by a plasmid gene. Althoughthis problem was solved by the introduction of methicillin and similar compounds that resistthe enzymatic hydrolysis, another enzyme, TEM β-lactamase, was reported in gram-negativebacteria in strains containing multiple-drug-resistant R plasmids that date from 1962 (12). Thisenzyme became widespread throughout the world, making penicillins with gram-negativeactivity, such as ampicillin, almost useless. (Methicillin and its relatives are inactive againstgram-negative bacteria because they are pumped out efficiently by the multidrug efflux pump;see below.) β-Lactamases are classified into several phylogenetic families. Class A includesboth the S. aureus and TEM enzymes, whereas Class C represents chromosomally codedenzymes (e.g., AmpC) that are present in many gram-negative bacteria. These two classes areboth similar to serine proteases in their mechanism, whereas Class B enzymes aremetalloenzymes that hydrolyze carbapenems efficiently.

In response to the spread of β-lactam resistance, various β-lactams were developed. Althoughthe first-generation cephalosporins, such as cephaloridine and cefazolin, were rapidlyhydrolyzed by both TEM and AmpC, both cephamycins (such as cefoxitin) and the third-generation cephalosporins containing an oxyimino side chain (such as cefotaxime) wereinitially reported to resist both types of enzymes. However, the former was inactive againstsome gram-negative bacteria such as Enterobacter and Serratia. Although the latter wascapable of killing these organisms, their introduction into the clinics was followed by theemergence of resistant strains that over-produced the chromosomal AmpC enzyme. In fact, theAmpC enzymes have very low KM values for these compounds, and the values of Vmax/KMwere quite high (13). The AmpC enzyme, however, needs to be induced, and the third-generation cephalosporins were effective against these bacteria simply because they wereineffective inducers of this enzyme. Thus, the third-generation cephalosporins selected forconstitutive mutants of ampC. Furthermore, strong expression of plasmid-coded AmpC hasbeen found recently in species that do not express the chromosomally coded ampC.

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Subsequently, fourth-generation cephalosporins (cefepime, cefpirome) that are more resistantto hydrolysis by the AmpC enzyme have been developed. However, continued selectivepressure resulted in the selection of plasmids that produced mutants of common enzymes, suchas TEM or its relative SHV, which can now hydrolyze third- and sometimes even fourth-generation cephalosporins. These enzymes are called ESBL (extended spectrum β-lactamases)(14).

Especially troublesome among the ESBL enzymes are those called CTX-M (15). The genescoding for these enzymes appear to have originated from the chromosome of an infrequentlyencountered gram-negative bacterium Kluyvera and have transferred to R plasmids. Thistransfer or mobilization unusually appears to have occurred many times, and consequently theenzyme rapidly became widespread among R-plasmid-containing pathogenic bacteria (16).

β-Lactams with a new nucleus, such as carbapenems (e.g., imipenem), still remain quiteeffective, but their use may eventually result in the increased prevalence of enzymes capableof hydrolyzing these compounds (17).

Another example of new resistance mechanisms arising through mutations of prevalentresistance genes is a plasmid-coded ciprofloxacin resistance gene (18). Here an aminoglycosideacetylase, AAC (6′)-Ib, has mutated to acetylate the amino group of ciprofloxacin.

Acquisition of Genes for Less Susceptible Target Proteins from Other SpeciesSequencing of the genes coding for the targets of penicillin, DD-transpeptidase or penicillin-binding proteins (PBPs), revealed that penicillin resistance among Streptococcuspneumoniae was due to the production of mosaic proteins, parts of which came from otherorganisms (19). We note that S. pneumoniae is an organism capable of natural transformationand may import foreign DNA. Interestingly, a similar mechanism of penicillin resistance wasalso found in another organism capable of natural transformation, Neisseria meningitidis.

An extreme case of this scenario is the generation of MRSA. MRSA strains contain a newmethicillin-resistant PBP, called PBP-2A or 2′, whose expression is often induced bymethicillin and other β-lactams. The gene for this new PBP is located in a large (30–60-kb)segment of DNA, which apparently came from an organism other than S. aureus (1,20) andalso contains other antibiotic resistance genes. S. aureus is not naturally transformable, and itis unclear how this horizontal transfer of a large DNA segment occurred.

Bypassing of the TargetVancomycin, a fermentation product from streptomycetes, has an unusual mode of action.Instead of inhibiting an enzyme, it binds to a substrate, the lipid-linkeddisaccharidepentapeptide, a precursor of cell wall peptidoglycan. Because of this mechanism,many assumed that it would be impossible to generate resistance against vancomycin.However, vancomycin resistance is now prevalent among enterococci, normal inhabitants ofour intestinal tract. Because enterococci are naturally resistant to β-lactams, aminoglycosides,macrolides, and tetracycline, these vancomycin-resistant strains of enterococci becomeprevalent in a hospital environment, colonize the patients, and cause infections that are difficultto treat.

Study of the resistance mechanism showed that the end of the pentapeptide, D-Ala-D-Ala, wherevancomycin binds, was replaced in the resistant strain by an ester structure, D-Ala-D-lactic acid,which is not bound by vancomycin (160). Production of this altered structure requires theparticipation of several imported genes.

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Preventing Drug Access to TargetsDrug access to the target can be reduced locally. It can be also reduced by an active effluxprocess. In gram-negative bacteria, the access can be reduced generally by decreasing the influxacross the outer membrane barrier.

Local inhibition of drug access—Tet(M) or Tet(S) proteins, produced by plasmid-codedgenes in gram-positive bacteria, bind to ribosomes with high affinity and apparently changethe ribosomal conformation, thereby preventing the association of tetracyclines to ribosomes(21). Plasmid-coded Qnr proteins, which have become more prevalent in recent years, arethought to protect DNA topoisomerases from (fluoro)quinolones (22).

Drug-specific efflux pumps—Drug resistance owing to active efflux was discovered withthe common tetracycline resistance protein TetA in gram-negative bacteria (23), whichcatalyze a proton-motive-force-dependent outward pumping of a tetracycine-Mg complex(24).

Nonspecific inhibition of drug access—In the laboratory where nutrient-rich mediumis used, β-lactams often select for porin-deficient mutants (25). However, the generallydecreased outer membrane permeability is somewhat detrimental to the bacterial growthbecause the nutrient influx is also reduced, and such mutants are not common among clinicalspecimens. Nevertheless, porin mutants are found in some species of Enterobacteriaceae(Enterobacter aerogenes, Klebsiella pneumoniae) as a means of last resort resistance to themore recent versions of β-lactams that withstand inactivation by common β-lactamases (26).Mutations within the coding sequences of the porin also have been reported, which possiblyreduce the permeation rates of bulky β-lactams without affecting those of smaller nutrientmolecules (26). The multidrug efflux pumps are discussed below.

SOURCES OF THE RESISTANCE GENESThere are so many resistance genes that rely on many different mechanisms. Where did theycome from?

Producing OrganismsAs described above, some of the aminoglycoside-resistant genes appear to be derived fromstreptomycetes producing these antibiotics. The genes coding for vancomycin resistanceappear to originate in a similar way. Resistance here requires the production of several newenzymes, and it is unlikely that the genes coding for these enzymes evolved in the few decadesafter vancomycin was introduced. Indeed, the genes in the vancomycin-resistant clinicalisolates of enterococci were found to be homologs of those found in the vancomycin-producingstreptomycetes, organized in exactly the same manner (27), an observation that leaves no doubton the origin of these resistance genes.

Microorganisms in the Environment, Especially SoilSome resistance genes are found in the chromosome of environmental bacteria. A classicalcase is the ampC gene in the environmental genera of Enterobacteriaceae, such asEnterobacter, Serratia, and Proteus, and in the soil organism P. aeruginosa. These genes donot show signs that they have been imported in the recent past, and it is revealing that theampC gene of the exclusive animal symbiont E. coli lacks the induction mechanism and thatthe pathogen Salmonella spp. lacks the ampC gene entirely. In this connection, examinationof a random collection of soil-dwelling strains of Streptomyces and their relatives showed that60% to 100% of them were resistant to several antibiotics tested, suggesting that antibiotic-resistant genes are abundantly present in this habitat (28). Although most antibiotics may be

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present in soil only at very low concentrations, the recent discovery of microorganisms thatutilize antibiotics as nutrients (29) is suggestive of the evolutionary origin of some antibioticdegradation (resistance) genes.

ASSEMBLY, MAINTENANCE, AND TRANSFER OF RESISTANCE GENESR plasmids often contain many resistance genes; they are maintained stably in the host strainsof bacteria and are transferred very efficiently to neighboring drug-susceptible cells. How doall these processes occur?

Assembly of Resistance Genes in R PlasmidsWe have seen that most drug resistance genes are effective when expressed from plasmids.Remarkably, many such genes are often present on a single R plasmid, so that multidrugresistance can be transferred to a susceptible bacterium in a single conjugation event. Whenthe R plasmids were discovered in Japan in the 1950s, many of them already containedresistance genes for aminoglycosides, tetracycline, chloramphenicol, and sulfonamides.Sequencing of many plasmids has now shown how this clustering occurred.

In the sequence of the early-generation R plasmids, we see that most of the resistance genesare components of transposons, which can deliver the genes to any piece of DNA. This is seenin the plasmid R100 (Figure 1).

Tn21 is a particularly remarkable example of large, complex, multiply composite transposons(31). Interestingly, it contains mercury resistance genes. The sulfonamide resistance andaminoglycoside resistance genes in Tn21 were assembled as described below.

The discovery that many resistance genes in R plasmids contain a unique 59-base 3′-sequencetag led to the discovery of a remarkable apparatus called an integron (32). An integron containsa gene coding for an integrase, which catalyzes the insertion of resistance genes at apredetermined site downstream from a strong promoter (Figure 2). Once integrated, theresistance gene becomes marked by the tag, so that it can easily become integrated into anotherintegron, perhaps containing a different set of resistance genes. In addition to this advantageof high mobility, the resistance genes when inserted into an integron become organized into asingle operon, with the same orientation of transcription under a strong promoter supplied bythe integron structure. In the example seen in Tn21, the integron already containing asulfonamide resistance gene sul1 and a truncated version of a multidrug efflux gene qacE hasintegrated an aminoglycoside resistance gene aadA1 at the specific integration site attI. Anintegron may contain up to eight resistance genes (33).

In addition, many of these integrons carry enzymatic machinery for transposing the entireintegron structure to other places. Furthermore, the integron is usually inserted into a largertransposon such as Tn21, which allows the entire array of the resistance genes to hop betweendifferent plasmids and between plasmid and chromosome. It is unclear how the modernintegrons evolved, but there is a rather similar mechanism that builds the assembly of manygenes in Vibrio cholerae (34).

Recently, many integron structures were found to be associated with a downstream structurecalled ISCR element, containing a putative transposase gene (35). It apparently functions inan unusual, open-ended transposition event and recruits various resistance genes and deliversthem close to the integron structure, resulting in the assembly of yet more resistance genes.

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Maintenance of R Plasmids in the Host CellsRecombinant plasmids derived from cloning vectors are frequently lost from the host cellseven when they exist in relatively high copy numbers. In contrast, most natural R plasmids areremarkably stable and are rarely lost during the multiplication of host cells, even when thecopy number is low. This is because R plasmids usually contain genes that ensure the correctpartitioning of copies to daughter cells (36). In addition, some natural plasmids contain the“killer” elements composed of a stable killer protein or mRNA and an unstable inhibitor proteinor antisense RNA, so that the loss of the plasmids will result in the death of the host cell (36).

Because of the presence of these regulatory networks, plasmids sharing a similar replication/partition machinery cannot stably coexist in the same host cell, and this phenomenon has beenutilized for the classification of plasmids into “incompatibility groups.” In plasmids of entericbacteria, this classification includes about two dozen such groups (37,38).

Cell-to-Cell Transfer of R PlasmidsR plasmids are not only stably maintained, but also usually transferred between bacterial cellsat a very high efficiency, in many cases approaching 100%. The molecular mechanism of thiscell-to-cell transfer has been studied mainly in E. coli and its relatives and in the plasmids ofincompatibility group FI (F or sex plasmid), P (RP4), and W. In recent years, the plasmidtransfer genes were found to be homologs of the virB genes of Agrobacterium tumefaciens,which transfer a piece of bacterial DNA into a plant nucleus, and several effector proteininjection systems of human and animal pathogens (39,40). These systems are now called TypeIV Secretion Systems. A specific sequence in the circular, double-stranded DNA of plasmidsis recognized, and a single-stranded cut is made by the VirD2/TraI enzyme (Figure 3), withthe enzyme protein remaining covalently attached to the 5′-end of the cut strand. This complexis sometimes called a relaxosome, as the nicking of one strand results in the relaxation ofsupercoiling of the plasmid DNA. The protein-(single-stranded) DNA complex is thenexported by the “mating-pair formation” complex, composed of about a dozen differentproteins (Figure 4). The export of relaxosomes, rather than protein-free DNA, explains theobservation that some Type IV Secretion Systems export pure proteins. Before this happens,however, the leading 5–-end of the exported DNA, with the VirD2/TraI protein attached, mustbe taken up by an ATPase (VirD4 or TraG). The latter is called a coupling protein because ofits role in coupling the export substrate to the export apparatus (Figure 4). Although one of theoriginal strands of the R plasmid DNA is transferred completely into the recipient cells, in thedonor cell this strand is replaced by a rolling-circle replication mechanism, so that the completeR plasmids now exist both in donor and recipient cells. One would suspect that the entry ofthe DNA of foreign origin would be recognized by the strain-specific restriction endonucleaseof the recipient cells. However, the initial DNA piece that enters is single stranded (althoughthe complementary strand will be synthesized soon) and thus may escape this mechanism. Inaddition, some R plasmids are equipped with functions that antagonize the attack by restrictionendonucleases (41).

Some R plasmids are very small and do not contain the genes needed for the construction of amating-pair formation complex. However, these plasmids can be mobilized by the transfergenes of other plasmids and can be transferred efficiently into recipient cells. Finally, it shouldbe emphasized that conjugational transfer of plasmids is not a novel mechanism invented formultidrug resistance; indeed, horizontal transfer of genes, to a large extent through theconjugational mechanism, has been the main mechanism used for the evolution of many groupsof bacteria (42).

Gram-positive bacteria also carry out cell-to-cell transfer of plasmids (43). In addition, a largetransposon of a special class, called conjugative transposons, play a prominent role in these

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bacteria (44). They use a λ-phage-like mechanism of transposition, which generates a circularDNA as an intermediate. This intermediate can insert at a predetermined location in the plasmidor chromosomal DNA, but it can also behave like a conjugative plasmid and can transfer itscopy into another bacterium by conjugation. Remarkably, in the transposons containing thetetracycline resistance gene, the transfer function is induced strongly by the presence oftetracycline. Thus, in these transposons, the drug resistance genes are not just passivepassengers but are a part of an integrated regulatory mechanism of conjugational transfer. Suchtransfer also occurs easily between distantly related organisms, for example between gram-positive and gram-negative bacteria (45).

MULTIDRUG EFFLUX PUMPSThe active efflux of drugs has been known to play a predominant role in the resistance to certainindividual drugs, such as tetracycline, as described above. Analysis of S. aureus strains thatwere resistant to multiple cationic bacteriocides and were causing hospital-acquired infectionsshowed, however, that these strains contained plasmids coding for a multidrug effluxtransporter QacA (or QacB), belonging to the Major Facilitator superfamily (MFS) (46), thefirst multidrug efflux pump identified in bacteria. Since then, multidrug efflux pumpsbelonging to various families have been discovered, and the contribution of such pumps tomultidrug resistance became clear (47,48). Structures and possible mechanisms of thesetransporters have been reviewed recently (49)

Multidrug Efflux Pumps Belonging to the Major Facilitator SuperfamilyMFS is one of the largest families of transporters and contains many important efflux pumps.

MFS Pumps with 14 transmembrane segments—QacA and QacB were the firstexamples of this class (46). These pumps actively extrude monocationic biocides and dyes,such as benzalkonium chloride, cetyltrimethylammonium bromide, and ethidium bromide, andin addition, QacA pumps out dicationic biocides, such as chlorhexidine and pentamidineisethionate. Each protein contains 14 transmembrane segments (TMSs) with several acidicamino acid residues in the transmembrane region. Comparison of these two protein sequencesand site-directed mutagenesis showed that the extrusion of dicationic compounds by QacA,but not by QacB, depended upon the presence of an aspartate residue in the TMS 10.

The expression of QacA pump is negatively regulated by the QacR repressor, and binding ofsome of the QacA substrates to QacR relieves this repression, resulting in the increasedexpression of the pump. Although QacA has not been crystallized, crystallographic studies ofthe QacR-inducer complex (50) gave us insights on how a binding site can accommodatediverse ligands. Thus, the QacR drug-binding pocket is large (1100Å3), and many drugs bindto one of the “minipockets” within this cavity. Unlike the binding of most ligands toconventional enzymes, the binding involves few hydrogen bonds and relies mostly on stackingand van der Waals interactions, with the charges of the ligands usually neutralized by acidicresidues. These features are similar to the binding of ligands to the human pregnane X receptor,a transcriptional regulator that binds to a number of xenobiotics (51). QacR can bind twoligands simultaneously to different minipockets of the binding site (52), and a given ligand canbe bound in different ways within the binding cavity (53). Another regulatory protein, TtgR,belonging to the same family as QacR but regulating a transporter in the Resistance-Nodulation-Division (RND) family (see below), was crystallized with several antibiotics andplant secondary metabolites (54). The binding pocket here is even larger (1500Å3), and someligands utilize the broad hydrophobic area of the pocket, whereas phloretin in its high-affinitymode binds to the deep valley within the pocket, utilizing several H-bonding interactions,consistent with the extremely wide range of ligands that regulate the expression of the cognateRND pump (54).

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Another multidrug transporter belonging to this branch is EmrB of E. coli, which confersresistance to uncouplers such as carbonyl cyanide m-chlorophenylhydrazone and to antibioticssuch as nalidixic acid and thiolactomycin (46,55). EmrB is coded for by a chromosomal gene,together with EmrA, a periplasmic adaptor protein that connects the pump to an outermembrane channel, TolC, so that the drugs can be exported directly into the medium, asdiscussed below in the section on RND family pumps.

MFS Pumps with 12 TMSs—The chromosomally coded NorA of S. aureus, in contrast,belongs to a branch with 12 TMS transporters (46). NorA produces resistance tofluoroquinolones, as well as to cationic dyes and cationic inhibitors including puromycin andtetraphenylphosphonium (56). The S. aureus chromosome contains at least two other homologsof NorA, NorB and NorC, which produce a similar phenotype (57). Because all these effluxpumps are inhibited by reserpine, sensitization of bacteria to substrate drugs in the presence ofreserpine can be used as an effective tool to assess the contribution of the efflux process toresistance. Thus, in about one-half of S. aureus strains isolated from the bloodstream ofpatients, efflux made a strong contribution to resistance, and more than half of these strainsoverexpressed the three chromosomally coded pumps just mentioned (58).

Some MFS pumps have been studied as model systems, although they have little relevance inthe clinic. LmrP of Lactococcus lactis pumps out cationic dyes, daunomycin, tetracyclines,and macrolides (59), and MdfA of E. coli can confer increased resistance to cationic dyes,chloramphenicol, and fluoroquinolones when overexpressed from plasmids in a mutant strainlacking the constitutive RND pump AcrB (60), although mdfA deletion produces no effect ondrug susceptibility (61). Study of the LmrP pump showed that fluorescent dyes are capturedfrom the inner leaflet of the bilayer, the pump thus acting as a “vacuum cleaner” of themembrane. The MdfA pump is remarkably versatile, for example, pumping out neutralsubstrates in an electrogenic manner while treating cationic substrates in an electroneutral way.Another unexpected finding was that the mdfA deletion mutants were hypersensitive to alkalinepH, presumably because MdfA functions as a K+/H+ antiporter (161). This is reminiscent ofthe similar function for a chromosomally coded “monodrug efflux pump,” TetB(L), of Bacillussubtilis (62).

Among MFS multidrug efflux pumps, the crystal structure was solved for EmrD of E. coli(63), which pumps out uncouplers, quaternary ammonium biocides, as well as sodiumdodecylsulfate (60). The central cavity, a likely substrate-binding site, is lined with side chainsof both an aliphatic and an aromatic nature.

Multidrug Efflux Pumps of the Small Multidrug Resistance FamilyEfflux pumps of a very different structure, belonging to the Small Multidrug Resistance (SMR)family (64), were first discovered coded on S. aureus plasmids and were subsequently foundto be coded also on the chromosomes of gram-negative bacteria. These proteins, which extrudecationic compounds such as quaternary ammonium biocides or ethidium, represent the smallesttransport proteins known, containing only four TMS in a 110-residue sequence in the EmrE ofE. coli. EmrE has been studied intensively (65). There is only one charged residue in thetransmembrane segments, Glu14, which has an unusually high pKa of about 8.5. When thetransporter encounters the substrate, this residue becomes deprotonated and instead binds thesubstrate, as shown with the transporter solution in detergents. Thus, the inward flux of proton(s) and the outward flux of the substrate appear to be coupled by the sharing of a commonbinding site. In terms of the three-dimensional structure, EmrE seems to exist primarily as asymmetrical dimer, according to the biochemical (66) and the electron cryomicroscopy data(67). Although much confusion ensued following the publications suggesting that the dimer is

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asymmetric, with one protomer facing the outside and the other facing inside, these papershave now been retracted as a result of calculations using a faulty software.

Multidrug Efflux Pumps of Resistance-Nodulation-Division FamilyTransporters of this family play, by far, a predominant role in the multidrug resistance of gram-negative bacteria.

Resistance-nodulation-division pump usually exists as a part of a tripartitecomplex—Efflux pumps of this superfamily (such as AcrB of E. coli and MexB of P.aeruginosa) (68) play an important role in producing multidrug resistance in gram-negativebacteria. This is because these pumps become associated with two other classes of proteins,the outer membrane channel exemplified by TolC of E. coli, belonging to the OMF (outermembrane factor) family of proteins (69) and the periplasmic “adapter” protein such as AcrAof E. coli and MexA of P. aeruginosa, classified into the MFP (membrane fusion protein)family (70) (Figure 5a). The association of these proteins was confirmed by chemical cross-linking (71) and by the isolation of the complex containing all three proteins (72). As wasemphasized already in 1996 (73), this construction allows the direct export of drugs into theexternal medium, rather than into the periplasmic space. This is a huge advantage because onceexported into the external space drug molecules must traverse the outer membrane barrier toreenter bacterial cells. Thus, these pumps work synergistically with the outer membrane barrier.Wild-type strains of most gram-negative bacteria are resistant to most lipophilic antibiotics(for E. coli, they include penicillin G, oxacillin, cloxacillin, nafcillin, macrolides, novobiocin,linezolid, and fusidic acid), and this “intrinsic resistance” was often thought to be caused bythe exclusion of drugs by the outer membrane barrier. Indeed, breaching the outer membranebarrier does sensitize E. coli cells to the drugs just mentioned (74). However, the inactivationof the major RND pump AcrB of E. coli makes the bacteria almost completely susceptible tothese agents [the minimal inhibitory concentration (MIC) of a lipophilic penicillin, cloxacillin,goes down from 512 μg/ml in the wild type to only 2 μg/ml (75)] even in the presence of theintact outer membrane barrier. Thus, the characteristic intrinsic resistance of gram-negativebacteria owes as much to the RND pumps as to the outer membrane barrier.

Some RND pumps show extremely wide substrate specificity. E. coli AcrB can pump out notonly most of the common antibiotics but also dyes, detergents, and even solvents (Table 1).Although AcrB cannot pump out aminoglycosides, there are homologs such as AcrD that carryout this function. Most gram-negative bacteria contain several chromosomal genes coding forsuch pumps, and their expression may become increased through regulatory responses ormutations, making the bacteria more resistant to practically all antimicrobial agents in onesingle step.

Finally, at least some RND pumps appear to capture the drugs either in the periplasmic spaceor from a location that is in equilibrium with the periplasm. This was predicted (73) becausecarbenicillin, which cannot cross the inner (cytoplasmic) membrane owing to the presence oftwo carboxylate groups, is still a good substrate for these pumps. This allows the pumps notonly to prevent the entry of drugs into the cytoplasm, but also to extrude agents, such as β-lactams, that have targets in the periplasm. Consistent with this concept, the substratespecificity of RND pumps is primarily determined by their large periplasmic, extramembranedomains, as shown by study of chimeric transporters (76) or mutants with altered substratespecificity (77). Importantly, the periplasmic capture mode allows the RND tripartite pumpsto collaborate synergistically with simple efflux pumps that extrude substrate drugs only intothe periplasm (78). A review describes pumps in P. aeruginosa (79).

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Regulation of RND pump expression—Expression of many RND pumps is repressedby specific repressor proteins (47,80). Sometimes, the pump expression is increased bysubstrates, for example, MexXY, an aminoglycoside efflux pump of P. aeruginosa, is inducedby its substrates (81). However, the mechanism of induction is more complex than with thatof QacA, described above, where the repressor QacR is inactivated by the binding of the inducerdrug. Thus, induction requires the inhibition of ribosomal function, which is not necessarilycaused by aminoglycosides (82), and apparently involves another protein (83).

With most other systems, the pump gene expression is not regulated by the substrates, and itis not known what ligands, if any, become bound to the local repressors. Thus, the efflux pumpoverproduction in clinical isolates occurs frequently as a result of mutation in the repressorgene, as with mexR mutations leading to overproduction of MexAB-OprM system in P.aeruginosa. A fraction of such MexAB-overproducing strains has no changes in the mexR geneand has mutations in the gene PA3721, the so-called nalC phenotype (84). Finally, the nalDgene, located upstream from the mexAB-oprM operon, also is involved in regulation (83). ThemexCD-oprJ operon is again under the control of the NfxB repressor, coded by an upstreamgene, and the pump overproducers have mutations in the repressor gene (85). The mexEF-oprN operon is exceptional in being regulated by an upstream activator, MexT. MexEF-OprN-overproducing strains do not contain mutations in the mexT gene, and instead have mutationsin PA2491, a gene coding for an enzyme-like molecule (86).

The expression of acrAB operon in E. coli is repressed by its cognate repressor AcrR, but thesmall effector molecules that bind to this repressor are not known. Instead, the transcription ofthe acrAB operon is mainly controlled by the AraC family global activators MarA, SoxS, andRob (47,80). MarA and SoxS are exceptionally small proteins containing only the DNA-binding domain, and thus, regulation occurs at the level of production of these proteins. TheMarA production is regulated by a repressor MarR, which becomes inactivated by salicylateand plumbagin. Interestingly, transketolase A, whose production is elevated under oxidativestress, binds to MarR and relieves its repression of MarA production (87). The level of activeSoxS repressor in the cell is determined by its own repressor SoxR, which contains Fe-S centersand becomes inactivated by superoxide. Rob is constitutively expressed and is also much largerthan MarA and SoxS, containing a domain outside the DNA-binding domain. Indeed, Robbecomes inactivated by binding substrate-like molecules, such as α,α′-dipyridyl (88), fattyacids, and bile salts (89).

Expression of some RND-type transporters, such as MdtBC (90,91), is regulated by a two-component system, BaeSR. Indole upregulates MdtBC as well as AcrD through BaeSR (92),although it is unclear if indole is directly sensed by the sensor kinase BaeS.

Plasmid-driven overproduction of SdiA, the soluble receptor for the quorum-sensing signalacyl-homoserine lactone, increased expression of AcrAB (93). However, growth of E. coli inthe presence of hexanoyl-homoserine lactone did not upregulate the acrAB operon (94).

Biochemical and crystallographic studies—Biochemical studies of the RND-typeefflux pumps were hampered by the fact that the pumps function as multiprotein complexesspanning two membranes. Nevertheless, the basic kinetic constants of the AcrB pump wererecently determined (162). When β-lactams are added to intact E. coli cells, they diffuse acrossthe outer membrane and, in periplasm, are either hydrolyzed by the periplasmic β-lactamaseor extruded by the AcrAB-TolC pump. Because the kinetic constants of the β-lactamase areknown, we can calculate the periplasmic drug concentration from the hydrolysis rate. Theefflux rate at that periplasmic concentration is obtained as the difference between the rate ofinflux (following Fick's first law of diffusion) and the hydrolysis rate. Data with nitrocefinsuggests saturation kinetics of efflux with a half-saturation concentration of ~5 μM and a

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turnover number about 10. Although nitrocefin did not show positive cooperativity, such abehavior was evident with cephalothin, cephamandole, and cephaloridine, which showed muchhigher K0.5 values than nitrocefin did. The positive cooperativity is consistent with thefunctionally rotating mechanism of AcrB function, described below.

Another biochemical approach used was the reconstitution of purified RND transporter intoproteoliposomes. Because most of the substrates of these pumps are lipophilic andspontaneously cross the bilayer, the assay used the movement of labeled phospholipid fromthe exporter-containing vesicle to acceptor vesicles that were devoid of proteins (95). Thisstudy showed that the AcrB pump functions as a proton antiporter and also showed that, amongthe substrates, conjugated bile salts had the highest affinity to AcrB. Another result was thatthe addition of AcrA increased very strongly the rate of transport (95). Although this wasinterpreted as the result of connection between vesicles mediated by AcrA, AcrA is essentialin producing the aminoglycoside pumping activity of reconstituted AcrD (96) where suchjuxtaposition of vesicles is not needed, and AcrA probably activates directly the pumpingactivity of AcrB.

Most important pieces of knowledge concerning the RND pump complex came fromcrystallography. Thus, the structure of the outer membrane channel TolC was solved in 2000(97,98) (shown in red in Figure 5a), followed by the structure of its P. aeruginosa homolog,OprM (99). These proteins exist as a tightly woven trimer, containing a single 12-stranded β-barrel traversing the outer membrane and a remarkably long (~70-Å) periplasmic extension ofthe channel in the form of long α-helical bundles. Murakami et al. (100) solved thecrystallographic structure of AcrB trimer in 2002, which was a first for a proton antiporter.The periplasmic portion is at least as large as the transmembrane portion, and the top of theperiplasmic domain, the TolC-binding domain, has a dimension that is similar to the tip of theα-helical bundle of TolC (Figure 5a,b). This was followed by the crystallographic elucidationof the structures of the central portion (about two-thirds) of the adaptor proteins MexA (101,102) and AcrA (103) (blue in Figure 5a). These are elongated proteins, as shown in 1999(104), and in crystals show a strong tendency to become packed side by side to form hexamersand heptamers. Thus, the structures of all three component classes of the tripartite assemblyare known, and it is possible to propose how these proteins are assembled together (105) (Figure5a). In such models, the coiled-coil domain of the adaptor is assumed to interact with the outermembrane channel protein, a hypothesis that is supported by biochemical data (106,107).Studies with chimeric constructs of AcrA homologs showed that the C-terminal domain, whosestructure is not known from crystallography, is essential for interaction with AcrB (108).Mutant studies also suggest that the domain close to this end of AcrA, containing the α + βstructure, is the main site of its interaction with the periplasmic domain of RND pumps(109).

The outer membrane component TolC and its homologs are first synthesized with the signalsequence and then are exported into the outer membrane. The periplasmic adaptors are eitherlipoproteins with the covalently bound lipid at its N terminus or contain a hydrophobic N-terminal helix and are thought to become bound to the outer surface of the inner membrane.The RND pump itself, as a classic example of polytopic inner membrane protein, requires thesignal-recognition particle and the SecY apparatus for insertion into the membrane (110). Theinteraction between the component proteins of the AcrB-AcrA-TolC complex was studied invitro by titration calorimetry (105).

Above, I mentioned that AcrB likely captures some of its substrates from the periplasm. Indeedthe AcrB trimer structure showed that there was a small opening (vestibule) between subunitsat the bottom of the periplasmic domain, close to the external surface of the membrane bilayer(100), and this led to the top of the large central cavity in the transmembrane domain (Figure

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5b). It was thus hypothesized that the drugs diffused through the vestibule into the centralcavity, where they were captured. Indeed, cocrystallization of AcrB with various drugsproduced crystals with drug molecules within this central cavity (111). However, there was noproof that the bound drugs were on their correct path to extrusion, and the extrusion pathwayswere not clear in the crystal structure. We have attempted to solve this problem by crystallizingAcrB mutants in which one of the residues putatively involved in proton translocation wasaltered; we assumed that such proteins would assume a conformation mimicking that of atransient intermediate during the process of drug extrusion (112). There was an extensiveconformational alteration in the transmembrane domain, but only minimal alterations wereseen in the periplasmic domain (112), which is likely the main site of substrate binding andextrusion (see above). The reason became clear from the papers describing the asymmetrictrimer structure of AcrB (113–115), especially as the Murakami group succeeded in solvingthe structure in which one of the protomers contained a substrate (minocycline or doxorubicin)(Figure 5c). Each protomer takes a conformation that is unique. Indeed the transmembranedomain of our mutant AcrB was similar in conformation to that of one of the protomers, calledthe extrusion protomer. However, extensive conformational changes in the periplasmic domainrequire complementary accommodation by the neighboring protomers (113–115). This wasnot possible in our construct, in which all protomers corresponded to the mutant protein. In thebinding protomer, the periplasmic domain encloses an expanded binding pocket containingseveral aromatic residues, and there is an open pathway between the binding pocket and thevestibule. In the extrusion protomer, the binding pocket becomes much narrower, and thepathway to the vestibule becomes closed (Figure 6). These crystallographic observationssuggest that the protomers within the trimeric AcrB go through a cyclic conformational change,from the open conformation through the ligand-bound one to finally the extrusion conformer,whose transmembrane domain shows signs of a disrupted network among proton-translocatingresidues. This cyclic change involves the opening and closing of the large external cleft in theperiplasmic domain, and the forced closing of this cleft by fast-acting disulfide cross-linkingagents stops the function of the pump instantaneously, thus providing biochemical support forthis hypothesis (116). A similar result was obtained with a somewhat different approach(117). More recently, we succeeded in producing a giant gene containing three copies of theacrB genes connected together by linker sequences and, by using this method, showedconclusively that inactivation of just one protomer in the trimeric complex completelyabolishes the pumping activity (118), a result supporting the functionally rotating mechanismmentioned above.

The binding pocket identified in the asymmetric structure (113–115) is large and flexible. Infact, the two substrates, which were cocrystalized with AcrB, minocycline, and doxorubicin,predominantly occupy somewhat different parts of the cavity; this explains the extremely widesubstrate specificity of this pump. The pocket in AcrB, which favors lipophilic substrates, hasa remarkably hydrophobic surface (Figure 7a). When AcrD, which pumps out basic andhydrophilic aminoglycosides (96), is homology modeled following the binding protomer ofAcrB, its hypothetical binding pocket is seen to contain many electronegative atoms (Figure7b). This observation increases our confidence that this is the genuine substrate binding site inthese transporters.

Finally, we note that inhibitors of RND multidrug efflux pumps have been developed (forexample, see Reference 121).

“Natural” substrates for multidrug efflux RND pumps—The idea that the multidrugefflux pumps may have other, more physiological functions than the extrusion of exogenouschemicals began with the finding that an MFS pump in B. subtilis, Blt, is actually a componentof an operon that detoxifies spermidine (122). Clearly the RND-type multidrug pumps havenot appeared in response to the widespread use of antibiotics, as most of these pumps are coded

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by chromosomal genes that are present in strains isolated long before the antibiotic era. ForAcrB of E. coli, its main physiological function is to protect the bacteria against bile salts,detergents that are abundant in the environment where E. coli lives, the intestinal tract ofvertebrates. This notion is supported by the observation that bile salts appear to have the highestaffinity, among substrates, to the AcrB transporter (95). The AcrAB-TolC system was reportedto be important in the pathogenicity of Salmonella spp. (123). However, this conclusion wasreached by orally infecting experimental animals with bacteria and seems less convincingbecause the infecting bacteria will obviously encounter bile acids where the AcrAB pump willbe needed for their survival. With plant pathogens, again RND pumps may be needed to protectthe bacteria against plant secondary metabolites. In Pseudomonas syringae, the inactivation ofone RND pump was reported to decrease the secretion of lipopeptide phytotoxins (124), butthe decrease was only modest (41% to 67%).

With soil organisms such as P. aeruginosa, it seems likely that most of the RND pumps areinvolved in excluding toxic compounds produced by other soil microorganisms. The reportsthat they are involved in the export of homoserine lactone autoinducers are not convincing, asthese compounds should diffuse across membranes spontaneously, and as the assay involvescells that are secreting these compounds and at the same time are exposed to the samecompound secreted by other cells. However, the loss of some RND pumps was shown to resultin the loss of invasiveness in the cell culture, through mechanisms that are unclear (125).

Contributions of RND efflux pumps to the resistance in clinical strains—RNDpumps are making major contributions both to the intrinsic and elevated resistance of clinicallyrelevant pathogens (48). For example, although fluoroquinoline resistance is often attributedto the mutational alteration of the target topoisomerases, high-level resistance seems to requirean added contribution by increased efflux (126). Fluoroquinolone resistance in P.aeruginosa is rapidly increasing; efflux makes a major contribution in most such cases (48).A significant portion of aminoglycoside-resistant clinical isolates of gram-negative bacteriawas previously classified as being caused by “decreased permeability.” These strains are nowknown to owe their resistance to increased active efflux (127,128). In view of the importantroles multidrug efflux plays in resistance, many scientists are concerned with the trend toincreasingly add disinfectants to household products like soaps, because such compounds mayselect for pump overproduction mutants (129).

RND transporters that pump out nondrug substrates—Some of the RND transportersare involved in the export of compounds other than the antimicrobials. The “Nodulation” partof its name came from the putative NodGHI proteins (130), which are now fused into one RNDprotein (131), involved in the secretion of “nodulation factors” or lipochitin oligosaccharidesby Rhizobium (132).

As mentioned above, typical multidrug exporters such as AcrB and MexB also export somesimple solvents. Organisms that resist high concentrations of solvents, such as Pseudomonasputida, contain RND transporters optimized for the efflux of solvents, some of which can alsopump out antimicrobials (133). Among the human proteins, the Niemann-Pick C1 diseaseprotein, a member of RND family, attracted attention because mutations in the protein causecholesterol accumulation in some organelles (134). Indeed, a close relative of this protein,Niemann-Pick C1-like protein, is involved in cholesterol transport in the intestinal epithelialcells (135).

There are many examples of RND transporters in the genome of M. tuberculosis. One of them(MmpL7) is involved in the export of complex, apolar lipid, phthiocerol dimycocerosate(136) and another one (MmpL8) in that of 2,3-diacyl-α,α′-trehalose-2′-sulfate, a precursor ofsulfatides (137).

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RND transporters also play a major role in resistance of gram-negative bacteria against toxicmetals (138). CzrA, an RND transporter in Ralstonia that pumps out Co2+, Ni2+, Cd2+, andZn2+, has been studied intensively, and an early successful reconstitution of an RND pump hasbeen achieved in this system (139).

Other Multidrug Efflux Pumps Energized by Ionic GradientsSeveral multidrug efflux transporters belong to the Multidrug and Toxin Extrusion (MATE)family (140). NorM of Vibrio parahaemolyticus, a protein with 12 transmembrane helices,pumps out fluoroquinolones and ethidium in exchange for the influx of Na+ (141). A similarpump was found in Bacteroides fragilis (142).

Multidrug Efflux Pumps of the ATP-Binding Cassette SuperfamilyTransporters of ATP-Binding Cassette (ABC) superfamily play a major role in the multidrugresistance of cancer cells (143). Their role in drug resistance, however, seems to be more limitedin bacteria (144). LmrA of L. lactis (145) is homologous to one-half of the mammalian MDR1protein. Although its overproduction in E. coli confers resistance to cationic dyes, daunomycin,and triphenylphosphonium, it does not seem to play a significant role in the drug resistance ofL. lactis. Nevertheless, biochemical studies of this pump led to a proposed mechanism for thecoupling of drug extrusion and ATP hydrolysis (146) and to the discovery that thetransmembrane domain of this pump can alone catalyze a proton-gradient-dependent flux ofdrugs (147). Studies were made on other ABC drug exporters of gram-positive bacteria, suchas BmrA of B. subtilis (148,149) and Sav1866 of S. aureus, whose X-ray crystallographicstructure (150) had a major impact in the studies of ABC transporters.

Another ABC transporter, MacB in E. coli, occurs together with the structural gene for aperiplasmic adaptor protein MacA and confers resistance to macrolides when overexpressed(151). In vitro reconstitution showed that the ATPase activity of MacB was stimulated byMacA, showing that the membrane fusion protein has functions other than the structural one(152).

MULTIDRUG RESISTANCE CAUSED BY ALTERED PHYSIOLOGICALSTATES

The antibiotic susceptibility of bacterial cells is affected by their physiological states. Oneimportant consequence of this phenomenon is the occurrence of “persister” cells. Thus, it wasdiscovered early that even high concentrations of antibiotics do not kill all of the bacterialpopulation, leaving behind a persister population that is genetically identical with thesusceptible cells (153). When biofilms were found more resistant to most antibiotics, therewere initial attempts to interpret this finding on the basis of a more limited diffusion of drugsthrough the biofilm structure. However, this mechanism cannot produce large increases inresistance. Although there are interesting data that link drug resistance in P. aeruginosabiofilms to the production of periplasmic β-(1–3)-glucans (154) and an efflux system (155), itis difficult, at least at present, to explain the extensive antibiotic resistance of biofilms on thebasis of alteration of all cells in the population.

It is therefore attractive to explain such resistance as the result of the presence of a large numberof persister cells in the biofilm population (153). The presence of persisters is now thought tobe an example of the strategy whereby bacteria naturally generate mixtures of phenotypicallydifferent populations, so that one of them can be advantageous to a changing environmentaldemand (156). Persisters limit the efficacy of antibiotic therapy, and we note that a recentsingle-cell study has identified an antibiotic-susceptible phase in the life cycle of typicalpersister cells (157).

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SUMMARY POINTS

1. Multidrug resistance in bacteria is often caused by the accumulation of genes, eachcoding for resistance to a single drug, on R plasmids. The assembly of resistancegenes on a single R plasmid is achieved by mechanisms provided by transposons,integrons, and ISCR elements. Integrons, for example, are especially powerful inproducing multidrug resistance because they assemble several resistance genes ina correct orientation and supply a strong promoter for their expression.Furthermore, the resistance gene once incorporated into an integron becomestagged, so that it could easily become a part of another integron.

2. Many of the resistance genes apparently have their evolutionary origins in theantibiotic-producing microbes, which must defend themselves from the antibioticsproduced. Many also come from the environmental organisms, especially soilmicroorganisms, which have been exposed to various antibiotics throughout theirevolutionary history. An alarming trend is the recent selection of mutants ofwidespread resistance genes that resulted in the broadening of the substrate rangeof the inactivated drugs, as in the extended-spectrum β-lactamases from thecommon TEM β-lactamase.

3. R plasmids are maintained extremely well and are often transferred efficientlyfrom cell to cell.

4. Another mechanism of multidrug resistance is the active pumping out of drugs bymultidrug efflux pumps. The RND superfamily pumps in gram-negative bacteriaare especially important because they are usually coded by chromosomal genesand can be overexpressed easily and because some of them can pump out most ofthe antibiotics currently in use.

5. In some gram-negative species, these mechanisms may become augmented by thedecrease in outer membrane permeability through mutations in porin genes.

6. Finally, persistence of pathogenic microorganisms may occur in an antibiotic-treated patient because they may get into a physiologically resistant state withoutany genetic changes.

FUTURE ISSUES

1. There is a continuing need to define the molecular details of the resistancemechanism. This may lead, for example, to useful inhibitors of multidrug effluxpumps or to inhibitors of the R plasmid transfer process.

2. There are also practical issues of preventing the further increase in multidrugresistant bacteria. Because the usage of antibiotics was the cause of selection ofthese bacteria, one logical conclusion is to minimize antibiotic usage. Indeed, thereis a striking correlation between the usage of β-lactams and the frequency ofoccurrence of penicillin-resistant pneumococci in the member countries ofEuropean Union (158).

3. Simultaneous administration of more than one agent (as is done with tuberculosis)may be considered. For example, an inhibitor of multidrug efflux pumps lowersthe MICs of various drugs strongly in gram-negative bacteria and may prevent theemergence of resistant organisms (121).

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AcknowledgmentsI thank colleagues for reading parts of this manuscript and for giving me the original figures for reproduction. Iapologize to authors whose studies could not be cited because of size limitations. The work in the author's laboratoryhas been supported by a research grant from U.S. National Institutes of Health (AI-09644).

Glossary

MRSA methicillin-resistant Staphylococcus aureus

Aminoglycosides bactericidal antibiotics that are active against those gram-negativebacteria, resisting most other antibiotics owing to their lowpermeability outer membrane

Transposons discrete DNA sequences that can move to another sites.“Composite” transposons contain antibiotic resistance genesbetween two simple transposons, i.e., IS elements

β-Lactams antibiotics, including penicillins, cephalosporins, andcarbapenems, which irreversibly inactivate DD-transpeptidasesinvolved in the cross-linking of bacterial peptidoglycan, oftencausing cell lysis

PBP penicillin-binding proteins, or DD-transpeptidases, which cross-link the newly made peptidoglycan and correspond to the targetsof β-lactam action

Porins proteins that produce, in the outer membrane of gram-negativebacteria, nonspecific diffusion channels that allow the influx ofmost of the effective antibiotics

Major Facilitatorsuperfamily (MFS) ofsecondary transporters

proton symporters or antiporters; some are involved in drugefflux, both single drug and multidrug

TMS transmembrane segment or the section of amino acid sequencethat traverses the membrane bilayer

Resistance-Nodulation-Division (RND)superfamily oftransporters

many are antibiotic/proton antiporters, often with very widesubstrate specificities

ABC a superfamily of ATP-dependent transporters, containing thetransmembrane domain and the ATP-binding cassette (ABC)domain

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Figure 1.Map of an early R plasmid R100. Tetracycline resistance gene tetA is in the transposonTn10, chloramphenicol acetyltransferase (cat) as a part of Tn9, and sulfonamide resistancegene sul1 and an aminoglycoside adenyltransferase gene aadA1 as a part of the large transposonTn21. The resistance genes are color coded, and the direction of transcription is shown by thearrows. Much of the blank areas outside the transposons are occupied by the transfer genes,needed for the formation of relaxosomes, the coupling factor, and the mating-pair formation.This figure is based on the nucleotide sequence deposited by (30) and GenBank sequence NC002134, submitted by G. Sempei and K. Mizobuchi. ΔqacE is a defective version of a genecoding for a multidrug efflux pump of Smr family (see text).

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Figure 2.Presumed mechanism of resistance gene capture by integrons. An integron contains the genefor site-specific integrase (blue) and the specific integration site attI (green). When theresistance gene 1 (red) in the circular cassette form containing the 3′-terminal 59-bp element(pink) is available, the gene is integrated at the attI site, regenerating a slightly altered attIsequence (now in green and pink). This can then accept the second resistance gene casette, andthe process can go on in this manner. The resistance genes, all in the same orientation, aretranscribed by the powerful promoter provided by the integron (Pant). From Reference 159with permission from Elsevier Ltd.

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Figure 3.Steps in T-DNA transfer in Agrobacterium tumefaciens (annotation on the left) and conjugativeplasmid DNA transfer in enteric bacteria (annotation on the right). From Reference 41 withpermission from Elsevier Ltd.

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Figure 4.A model of the mating-pair formation complex with the coupling protein (VirD4). The proteinsare named after the VirB components in Agrobacterium tumefaciens, but homologs for mostof these proteins are found in the conjugative R plasmid transfer systems. Number n denotesthe product of virBn gene. The virD4 product is shown as D4. From Reference 39 withpermission from Elsevier Ltd.

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Figure 5.(a) The model of the AcrB-AcrA-TolC tripartite complex that exports drugs directly into themedium. The transmembrane domain of the AcrB pump trimer is embedded in the cytoplasmicmembrane, whereas its periplasmic domain is connected to the TolC channel through a numberof periplasmic AcrA linker proteins. From Reference 105 with permission from Elsevier Ltd.(b) AcrB trimer. Each protomer is shown in a different color. The large central cavity (thickblack lines) is connected to the periplasm through vestibules (thick dotted lines) betweenprotomers. The proximal portion of the structure was cut away to reveal the presence ofvestibule. Drawn by using PyMol with Protein Data Bank coordinate file 1OYE. (c) Theperiplasmic domain of the asymmetric AcrB trimer viewed from the top. The conformation ofeach protomer is characteristic, with open or closed external clefts. A drug molecule is seenbound to the binding protomer. From Reference 113 with permission from Nature PublishingGroup.

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Figure 6.Cut out view of the binding protomer with the bound minocycline (in a ball-and-stickrepresentation in green) (a) and the extruding protomer (b), both from Protein Data Bank file2DRD. Molecular graphics images were produced using the UCSF Chimera package from theResource for Biocomputing, Visualization, and Informatics at the University of California, SanFrancisco (supported by NIH P41 RR-01081) (119). The wide passageway from the externalsurface to the binding site (dashed arrow) seen in (a) appears to be completely closed in (b).The dashed lines show the approximate limits of the membrane bilayer. Modified after (120)with permission from Elsevier. The inset shows the location and direction of the clipping planeas well as the direction of the view in (a). The binding, extrusion, and access protomers areindicated.

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Figure 7.Substrate binding pockets of AcrB and a homology-modeled AcrD. (a) Residues facing thebound minocycline (in a ball-and-stick model), i.e., residues 177, 178, 275–278, 610, 612, 615,620, 626, and 628 of the binding protomer of AcrB in Protein Data Bank file 2DRD are shownin space-filling models. The walls are remarkably hydrophobic, as seen for example in the areacovered by the yellow ellipse. (b) Hypothetical structure of the corresponding region in AcrD.AcrD structure was built by homology modeling, using the binding protomer of AcrB as thetemplate. The area covered by the ellipse in (a) is now seen to be studded with oxygen atoms.The oxygen atoms shown by the arrowheads belong to the side chains of, from top, Serreplacing Phe617 of AcrB, Tyr replacing Ile277, and Thr replacing Phe611. Molecular graphicsimages were produced using the UCSF Chimera package from the Resource for Biocomputing,Visualization, and Informatics at the University of California, San Francisco (supported byNIH P41 RR-01081) (119).

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Tabl

e 1

Prop

ertie

s of s

ome

Res

ista

nce-

Nod

ulat

ion-

Div

isio

n (R

ND

) mul

tidru

g ef

flux

pum

psa

Org

anis

mR

ND

pum

p

Ass

ocia

tes w

ith:

Subs

trat

esb

Reg

ulat

orM

FPO

MF

Esch

eric

hia

coli

Acr

BA

crA

TolC

AC

, BS,

CM

, CP,

CV

, EB

, FA

, FQ

, ML,

NO

, OS,

PN

, SD

S, T

C, T

M, T

R, T

XA

crR

, Mar

A, S

oxS,

Rob

, Sdi

A

E. c

oli

Acr

DA

crA

TolC

AG

, DC

, FU

, NO

Bae

R

Pseu

dom

onas

aer

ugin

osa

Mex

BM

exA

Opr

MA

C, A

G, C

M, C

P, C

V, E

B, M

L, N

O, O

S, P

N, S

DS,

SF,

TC

, TM

, TR

Mex

R, N

alC

(PA

3721

), N

alD

P. a

erug

inos

aM

exD

Mex

CO

prJ

CM

, CP,

FQ

, ML,

NO

, TC

, TR

Nfx

B

P. a

erug

inos

aM

exF

Mex

EO

prN

CM

, FQ

, TC

Mex

T

P. a

erug

inos

aM

exY

Mex

XO

prM

AG

, FQ

, ML,

TC

Mex

Z

a Prop

ertie

s of w

ell-s

tudi

ed p

umps

are

show

n to

geth

er w

ith th

e pe

ripla

smic

ada

ptor

pro

tein

(MFP

) and

the

oute

r mem

bran

e ch

anne

l (O

MF)

they

inte

ract

with

. Sou

rces

for t

he d

ata

can

be fo

und

in (4

7,79

) as

wel

l as i

n th

e te

xt.

b Abb

revi

atio

ns fo

r sub

stra

tes:

AC

, acr

iflav

ine;

AG

, am

inog

lyco

side

s; B

S, b

ile sa

lts; C

M, c

hlor

amph

enic

ol; C

P, c

epha

losp

orin

s; C

V, c

ryst

al v

iole

t; D

C, d

eoxy

chol

ate;

EB

, eth

idiu

m b

rom

ide;

FA

, fat

ty a

cids

;FQ

, flu

oroq

uino

lines

; FU

, fus

idic

aci

d: M

L, m

acro

lides

; NO

, nov

obio

cin;

OS,

org

anic

solv

ents

; PN

, pen

icill

ins;

SD

S, so

dium

dod

ecyl

sulfa

te; S

F, su

lfona

mid

es: T

C, t

etra

cycl

ines

; TM

, trim

etho

prim

; TR

,tri

clos

an; T

X, T

riton

X-1

00.


Development of Multiple Antibiotic Resistance in Microbes (Microbial Genetics)

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